PROJECT SUMMARY The ability to sense and respond to diverse and dynamic changes in environmental conditions is crucial for bacterial fitness. Numerous signals must be integrated by cellular regulatory systems in order to promote optimal responses to those changing conditions. Salmonella is an important model organism for understanding genetic regulation and bacterial pathogenesis. A requisite for Salmonella to cause disease is the direct injection of effector proteins into host cells via a Type Three Secretion System (T3SS) encoded on Salmonella Pathogenicity Island 1 (SPI1). SPI1 has become a paradigm for understanding how bacteria use diverse global regulatory systems to respond to numerous environmental signals. Our long term goal is to understand at a systems level how the signals that control SPI1 are integrated at transcriptional, post- transcriptional and post-translational levels to allow the appropriate timing and magnitude of SPI1 gene expression. We have formulated a new model for the SPI1 regulatory circuit in which the three AraC-like regulators HilD, HilC, and RtsA act in a complex feed-forward regulatory loop to control expression of hilA, encoding the direct regulator of the SPI1 structural genes. Much of the regulatory input is integrated at the level of HilD, including at hilD mRNA translation or stability, while additional regulatory systems bypass HilD to directly control hilA. We hypothesize that multiple signals control translation of central SPI1 virulence regulators, hilD and hilA, and that much of this regulation is mediated by small RNAs (sRNAs). Indeed, our extensive preliminary data have revealed sRNAs that regulate both hilD and hilA translation or mRNA stability, and computational predictions suggest numerous additional sRNAs that act on these targets. This includes regulation via the unique 300 nucleotide 3' untranslated region of the hilD mRNA. The specific aims of this proposal are to: 1. Identify and classify sRNAs that base pair with mRNAs encoding major SPI1 virulence regulators, hilA and hilD. We have developed a series of reporter fusions that allow us to test regulation by sRNAs and precisely determine their site(s) of action. 2. Determine how sRNAs regulate SPI1 at a molecular level. Biochemical and genetic experiments will define sRNA-mRNA base pairing interactions, as well as elucidate specific regulatory mechanisms. 3. Elucidate the impacts of sRNAs on Salmonella physiology and virulence regulation. Expression analysis and tests of epistasis will place the identified sRNAs into the overall physiological framework regulating SPI1 expression. Single cell analysis will test the effects of sRNAs on the dynamics of SPI1 induction or shut-down. We will integrate this information into our existing mathematical model to expand our overall understanding of signal integration. The regulation of the SP1 T3SS serves as a paradigm for the integration of host environmental signals to control a complex virulence phenotype. Our work to uncover the molecular mechanisms controlling this system serves as a detailed model for other systems and will ultimately be critical to our understanding of this important pathogen.